Stable preparations of Acanthamoeba myosins IA and IB have been obtained which, for the first time, appear to be suitable for extensive physical chemical characterization of the differences between the phosyphorylated (active) and non-phosphorylated (inactive) forms of the enzyme. Acanthamoeba myosin II contains three phosphorylation sites on each heavy chain that can be phosphorylated by a partially purified myosin II heavy chain kinase. In each case, a serine is phosphorylated. The three sites lie within a segment of about 3000 daltons very near the end of the tail of the heavy chain and about 100,000 daltons away from the ATPase site that they regulated. So far, only two of the three sites have been shown to be phosphorylated in vivo but this is sufficient to inactivate the enzyme completely. Actin-activatable myosin II (dephosphorylated form) shows stimulation by Ca++ at 3mM Mg++ but not at 4mM Mg++. It is inactive at less than 3 mM Mg++. Enzymatic activity shows indications of cooperative myosin-myosin interactions but there seems to be no correlation between enzymatic activity and filament formation.